In general, cancer refers to the uncontrolled proliferating of cells. Lumps of such cells are classified into malignant tumors that lead to the death of an individual, and benign tumors that do not cause death. It can sometimes be difficult to accurately classify tumors. Most cancers are induced by cancer-triggered genes or mutations in cancer-inhibiting genes. The proliferation of cells regulates cell differentiation, a signal transduction system controlling the cell cycle and signal transduction between cells. When the regulation is abnormally performed, the cells cannot be differentiated and ceaseless proliferation is induced. This process is called carcinogenesis. If a method of correcting the abnormal regulation of the cancer-triggered protein is found, such a method would possibly be the most effective anti-cancer medicine without nonspecific effects.
Currently, cancer patients are treated through surgery, chemotherapy and radiation therapy. Substantially, complex cancer treatments combining such therapies and laser surgery are used. Chemotherapy is used prior to other treatments to reduce pain during treatment or to prevent the spread of cancer. Many anti-cancer agents have been discovered through much research, and most of them focus on selectively killing vigorously dividing cells. However, such agents cannot be used for long term treatment due to the side effects of killing normal cells such as immune cells or hair follicle cells.
Most anti-cancer agents destroy cancer cells by suppressing the synthesis of nucleic acids or interrupting cell metabolisms directly bound to the nucleic acids. Generally, anti-cancer agents cannot selectively attack malignant tumors. Anti-cancer agents attack and damage vigorously dividing normal cells such as hair, haemopietic cells in bone marrow, mucosal gastric cells and intestinal mucosa cells to cause side effects such as depilation, anemia, leukopenia, thrombocytopenia, gastric and intestinal mucosal damage. Besides causing side effects, conventional anti-cancer agents are not effective against tumors that spread to other tissues of the body.
Anti-cancer agents are classified into cytotoxic anti-cancer agents and hormonal anti-cancer agents. Examples of cytotoxic anti-cancer agents include Altretamine and Busulfan, and examples of hormonal anti-cancer agents include Tamoxipan and Toremifene. Cytotoxic anti-cancer agents transform into a material having a positive charge and covalently bind to a substance having plentiful electrons and ions such as nucleic acids, proteins and amino acids, thereby suppressing the synthesis of DNA and RNA. Hormonal anti-cancer agents bind to receptors of estrogen, thereby suppressing proliferation of estrogen dependent breast cancer cells.
In gene therapy, to treat genetic diseases and cancers caused by gene mutations, genes are directly inserted into cells having genes affected by a disease so that the function of the cells is normalized by expressing the inserted genes. The gene therapy can be widely used to prevent various diseases or to reinforce treatment by inserting a specific gene into a body cell and granting a new function to the body cell.
The most important factor in the treatment of diseases using gene therapy is that the inserted gene be successfully delivered to the nucleus of the target cell and that the gene be expressed strongly. The gene enters the target cell through endocytosis and is transported into the nucleus to be expressed. The gene can be inserted using a carrier such as a liposome since most DNAs are destroyed when entering the cell. However, most of the liposomes are also destroyed when entering the nucleus, thereby decreasing the transporting efficiency.
A virus capable of infecting a human can be treated using gene therapy because the virus effectively inserts exogeneous genes into the human body. Specifically, the gene can effectively be transported and expressed by inserting the gene for the gene therapy into the DNA of the virus using gene recombination and infecting human body with the recombinant virus, which can be mass produced in vitro. An adenovirus can be effectively used for the gene therapy by using a special mechanism of transporting the gene into the nucleus of the target cell with a high efficiency. In addition, retroviruses are being used in more than 50% of internationally permissible clinical trials (Wiley Online Library—The Journal of Gene Medicine). Retroviruses are effective for gene therapy when inserted into cell chromosomal DNA to allow long term expression of the desired protein.
One of the features of tumor cells is chromosomal abnormality such as aneuploid or tetraploid due to structural instability of chromosomes, or multinucleation. When spindle fiber check points lose the ability to regulate cell division, DNA synthesis is known to be carried out through endoreduplication (Jallepalli P V and Lengauer C. (2001). Nat. Rev. Cancer, 1, 109-117). Mad2 is an important protein delaying cell division until all spindle fibers are attached to the centromere (Hardwick K G, Johnston R C, Smith D L, Murray A W. J. Cell Biol. 2000 Mar. 6; 148(5):871-82). A recent article reported that while a partial loss of Mad2 promotes multinucleation, resulting in cancer (Dobles M, Liberal V, Scott M L, Benezra R and Sorger P K. 2000. Cell, 101, 635-645), and a complete loss of Mad2 using siRNA generates a signal inducing death of cancer cell line (Michel L, Diaz-Rodriguez E, Narayan G, Hernando E, Murty V V, Benezra R. Proc Natl Acad Sci USA 2004; 1001:4459-64; Kops G J, Foltz D R, Cleveland D W., Proc Natl Acad Sci USA. 2004 Jun. 8; 101:8699-704). P31comet is a protein that regulates Mad2 by interacting with Mad2 bound to APC/CCdc20 when a mitosis check point is activated during the metaphase of cell division (Habu T, Kim S H, Weinstein J and Matsumoto T. 2002. EMBO J., 21; 6419-6428). P31comet also promotes the activity of APC/C suppressed by Mad2. These features show that p31comet functions against Mad2 (Xia G, Luo X, Habu T, Rizo J, Matsumoto T, Yu H., EMBO J. 2004 Aug. 4; 23(15):3133-43).
Therefore, the inventors of the present invention conducted research based on the idea that overexpression of p31comet in cancer cells can completely inhibit Mad2, thereby killing the cancer cell. The p31comet gene was introduced into an adenovirus vector and a retrovirus vector to effectively express p31comet in a cancer cell and viruses including p31comet were produced. The produced viruses were injected into various cancer cells and the expression of p31comet was identified. A fluorescence protein is designed to be expressed together with p31comet to distinguish expressed cancer cells from non-expressed cancer cells. In the cells infected with the retrovirus or adenovirus including p31comet, it was found that the fluorescent cells are dead or the growth of the fluorescent cells stops after a certain period of time.